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Application of FNLS-sABE system in creation of rice herbicide-resistant material

A herbicide and rice technology, applied in the field of application of the FNLS-sABE system in the creation of rice herbicide-resistant materials, can solve the problems of mutations containing other sites, high identification costs, and low editing efficiency

Active Publication Date: 2020-04-03
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cysteine ​​(C) at position 2099 in rice OsACC1 corresponds exactly to the cysteine ​​at position 2088 in the above-mentioned weeds, and it is mutated to arginine (R), that is, C2099R can make rice produce ACCase and inhibit weeding Herbicide resistance, C2099R can be obtained by single base editing technology, but most of the base editing systems reported to create ACCase-inhibiting herbicide-resistant rice with mutation C2099R have low editing efficiency, and the mutants produced not only contain OsACC1 2099 Mutations at this site, and some also contain mutations at other sites, the cost of molecular identification of mutants is high

Method used

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  • Application of FNLS-sABE system in creation of rice herbicide-resistant material
  • Application of FNLS-sABE system in creation of rice herbicide-resistant material
  • Application of FNLS-sABE system in creation of rice herbicide-resistant material

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Effect test

Embodiment 1

[0095] Embodiment 1, a kind of ACCase inhibits the preparation method of herbicide-resistant rice

[0096] 1. Design and Construction of Recombinant Expression Vectors

[0097] 1. Design of recombinant expression vector

[0098] The present invention uses four types of adenine base editors to prepare ACCase-inhibiting herbicide-resistant rice. The schematic diagrams of the recombinant expression vectors of four different types of adenine base editors are as follows figure 1 shown. details as follows:

[0099] sABE system: add the bpNLS nuclear localization signal before the ecTadA element in the ecTadA&ecTadA*&Cas9n base editing system, and add the bpNLS nuclear localization signal after the Cas9n element. The amino acid sequence of the bpNLS nuclear localization signal is as follows: KRTADGSEFEPKKKRKV (SEQ ID NO: 7). This design type is denoted as bpNLS-bpNLS.

[0100] FNLS-sABE system: Add 3*Flag1&1*NLS1 nuclear localization signal before the ecTadA element in the ecTad...

Embodiment 2、A7

[0135] ACCase of embodiment 2, A7>G7 mutant ACCase suppresses herbicide resistance detection

[0136] 1. Herbicide spraying experiment

[0137] Randomly select 15 rice positive T0 seedlings with A7>G7 mutations and 15 rice positive T0 seedlings (referred to as WT seedlings) without A7>G7 mutations in the 15 strains obtained in step 3 of Example 1, and follow the steps 1-4 respectively To operate:

[0138] 1. When the rice positive T0 seedlings grow to about 15-20 cm in the rooting medium, open the lid of the culture dish, put in clean water to harden the seedlings, and culture them under light at 25°C for 5 days.

[0139] 2. After the seedling hardening, take out the T0 seedlings from the medium, wash the residual medium in the roots with clean water, send them to the greenhouse and transplant them into small pots, and transfer them to large pots for cultivation after 15 days.

[0140] 3. At the 6-10 leaf stage, randomly select three rice positive T0 seedlings with A7>G7 mut...

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Abstract

The invention discloses an application of an FNLS-sABE system in creation of a rice herbicide-resistant material. The FNLS-sABE system comprises a nuclear localization signal A and a nuclear localization signal B, and the nuclear localization signal A comprises a 3*Flag1 tag protein and an NLS1 protein; and the nuclear localization signal B comprises the NLS1 protein. The invention also disclosesa preparation method of rice resistant to an ACCase inhibiting herbicide. The method comprises the following steps: expressing esgRNA, adenine deaminase, Cas9 nuclease, the nuclear localization signalA and the nuclear localization signal B in receptor rice. The adenine deaminase, the Cas9 nuclease, the nuclear localization signal A and the nuclear localization signal B can mutate the 7th site ofan OsACC1 gene target sequence in a receptor rice genome from A to G under the guidance of the esgRNA so as to obtain rice resistant to the ACCase inhibiting herbicide.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of the FNLS-sABE system in creating rice herbicide-resistant materials. Background technique [0002] CRISPR-Cas9 technology has become a powerful genome editing method and has been widely used in many tissues and cells. The CRISPR / Cas9 protein-RNA complex is positioned on the target by the guide RNA (guide RNA), cuts and generates a DNA double-strand break (dsDNA break, DSB), and then the organism will instinctively initiate a DNA repair mechanism to repair the DSB. There are generally two repair mechanisms, one is non-homologous end joining (NHEJ), and the other is homologous recombination (homology-directed repair, HDR). Usually NHEJ accounts for the majority, so the random indels (insertions or deletions) generated by the repair are much higher than the precise repair. For precise base substitution, the application of HDR to achieve precise base subs...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N9/22A01H5/00A01H6/46
CPCC12N15/8213C12N15/8274C12N9/22C12N9/78C12Y305/04002C12N15/113C12N2310/10C12N2310/20
Inventor 杨进孝王飞鹏张成伟李璐
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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